Elsevier

Journal of Chromatography B

Volume 827, Issue 1, 15 November 2005, Pages 119-126
Journal of Chromatography B

Susceptibility of actin to modification by 4-hydroxy-2-nonenal

https://doi.org/10.1016/j.jchromb.2005.02.025Get rights and content

Abstract

4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, reacts with histidine, lysine or cysteine residues of proteins to form hemiacetal Michael adducts and thus interferes with the functions of the proteins. Here we undertook to identify HNE-modified proteins in the target organ of a ferric nitrilotriacetate (Fe-NTA)-induced renal carcinogenesis model with histidine-specific HNEJ-2 antibody. Immunoaffinity column separation and sequencing identified one of the major modified proteins as actin. To further explore the characteristics of actin as an HNE acceptor, we produced four novel monoclonal antibodies against HNE-modified keyhole limpet hemocyanin. All these antibodies (HNEJ-1, 3–5) recognized histidine adducts, but were different from HNEJ-2 in recognizing lysine and cysteine adducts to some extent. Actin, albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), metallothionein and superoxide dismutase were treated in vitro with HNE and evaluated with these antibodies. The results revealed that actin was most sensitive to HNE modification and metallothionein most resistant. Furthermore, the residue-specificity of GAPDH was in accord with that shown by our recent mass spectrometry data. Immunohistochemistry with the antibodies revealed cytoplasmic staining with or without nuclear staining in the renal proximal tubules after Fe-NTA administration. The results suggest that actin is a major target protein for HNE modification in vivo, and that our monoclonal antibodies are useful for evaluating the HNE adducts produced.

Introduction

Several lines of evidence indicate that oxidative modification of proteins and the subsequent accumulation of the modified proteins occur in cells and tissues during aging, carcinogenesis and various pathological conditions, including atherosclerosis, autoimmune diseases and neurodegenerative diseases [1], [2]. The important chemicals that give rise to the covalent modification of proteins may include reactive aldehydes, such as keto aldehydes, 2-alkenals and 4-hydroxy-2-alkenals [3], [4]. These reactive aldehydes are considered to be important mediators of cellular damage due to their ability to covalently modify biomolecules in ways that may disrupt important molecular structures, cause genetic alterations or activate certain signal pathways [4].

4-Hydroxy-2-nonenal (HNE), one of the reactive aldehydes, is a major product of lipid peroxidation [4], [5], [6] and is believed to be largely responsible for the cytopathologic effects observed during oxidative stress. HNE exerts these effects because of its strong reactivity with biomolecules, especially cysteine, histidine and lysine residues of proteins, producing cyclic hemiacetal Michael adducts [4].

Administration of an iron chelate, ferric nitrilotriacetate (Fe-NTA), provides an intriguing model of iron-mediated oxidative stress-induced carcinogenesis: intraperitoneal administration of this chemical causes renal proximal tubular damage as a consequence of the Fenton reaction that ultimately leads to up to 90% incidence of renal cell carcinoma in rodents [7], [8], [9]. This model is characterized by (1) high incidence of pulmonary metastasis and peritoneal invasion, (2) an increase in molecules covalently modified by reactive oxygen species [10] in the kidney during carcinogenesis, and (3) reduction of not only acute renal tubular damage but also tumor incidence [11] by pretreatment with a lipophilic antioxidant, α-tocopherol. We previously reported an increase in a variety of oxidatively modified molecules such as 8-oxoguanine [12], thymine-tyrosine cross-links [13], saturated and unsaturated mutagenic aldehydes including HNE and malondialdehyde (MDA), and HNE- and MDA-modified proteins [6], [14], [15] in the kidney after Fe-NTA administration. Recently, we identified the p15INK4B and p16INK4A tumor suppressor genes [16], [17] and the annexin 2 gene [18] as target genes in this model.

In the present study, we attempted to identify major HNE-modified proteins in the kidneys of rats after a single administration of Fe-NTA, and identified actin as one of them. Furthermore, we evaluated the characteristics of actin as an HNE acceptor by comparing its immunoreactivity with those of selected proteins using five different monoclonal antibodies (mAbs) produced against HNE-modified keyhole limpet hemocyanin (KLH).

Section snippets

Materials

trans-4-Hydroxy-2-nonenal was prepared as previously described [19] by acid treatment (1 mM HCl) of HNE diethylacetal, which was synthesized according to the procedure of De Montarby et al. [20]. The concentration of the HNE stock solution was determined by measurement of UV absorbance at 224 nm [21]. Ferric nitrate enneahydrate was from Wako (Osaka, Japan); nitrilotriacetic acid (NTA) disodium salt was from Nacalai Tesque Inc. (Kyoto, Japan). Nα-acetyl-l-histidine, Nα-acetyl-l-lysine,

Identification of a major HNE-modified protein in the kidney after Fe-NTA administration

SDS–PAGE analysis of the sample after HNEJ-2 column separation revealed two major bands of ∼33 and ∼46 kilodaltons (kDa) (Fig. 1A). The band of ∼46 kDa was subjected to amino acid sequencing. The obtained partial sequence was DLYANTVLSG, which was identified as an actin fragment using NCBI database analysis (http://www.ncbi.nlm.nih.gov/BLAST). Immunoprecipitation and Western blot analysis confirmed the results (Fig. 1B). Analysis of actin in the kidney 3 h after Fe-NTA administration revealed

Discussion

In the present study, we identified for the first time one of the major HNE-modified proteins as actin in an oxidative stress-induced carcinogenesis model of rats by the use of HNEJ-2 mAb affinity chromatography. The identification was confirmed by immunoprecipitation and Western lot analyses (Fig. 1B and C). Interestingly, HNE induced fragmentation of actin, and at least two of the fragments were modified with HNE, suggesting that protein inactivation by free radicals had occurred.

Oxidative

Acknowledgements

We thank Ms. Waka Kawaguchi for excellent technical assistance. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan and a Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare of Japan.

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